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Comparison between electroporation and polyfection in pig sperm: efficiency and cell viability implications

Published online by Cambridge University Press:  13 September 2018

Zigomar da Silva Open the ORCID record for Zigomar da Silva [Opens in a new window] ,
Andressa Pereira de Souza ,
José Rodrigo Claudio Pandolfi ,
Francisco Noé da Fonseca ,
Carlos André da Veiga Lima-Rosa  and
Mariana Groke Marques
Zigomar da Silva
Affiliation:
Santa Catarina State University. College of Agriculture and Veterinary – CAV, Av. Luiz de Camões, 2090, Conta Dinheiro, CEP: 88.520–000, Lages City, SC, Brazil
Andressa Pereira de Souza
Affiliation:
Santa Catarina State University. College of Agriculture and Veterinary – CAV, Av. Luiz de Camões, 2090, Conta Dinheiro, CEP: 88.520–000, Lages City, SC, Brazil Concordia College. Anita Garibaldi Street, 3185, Primavera 89701-130, Concórdia, SC, Brazil
José Rodrigo Claudio Pandolfi
Affiliation:
Brazilian Agricultural Research Corporation, Embrapa Swine and Poultry, 13 BR-153, Km 110, Tamandua District, Post Box: 321 CEP: 89715-899, Concórdia, SC. Brazil
Francisco Noé da Fonseca
Affiliation:
Brazilian Agricultural Research Corporation, Embrapa Swine and Poultry, 13 BR-153, Km 110, Tamandua District, Post Box: 321 CEP: 89715-899, Concórdia, SC. Brazil
Carlos André da Veiga Lima-Rosa
Affiliation:
Santa Catarina State University, College of Southern Region – Ceres, Street Cel. Fernandes Martins, 270, Progresso, CEP: 88.790-000, Laguna City, SC, Brazil
Mariana Groke Marques*
Affiliation:
Brazilian Agricultural Research Corporation, Embrapa Swine and Poultry, 13 BR-153, Km 110, Tamandua District, Post Box: 321 CEP: 89715-899, Concórdia, SC. Brazil
*
Author for correspondence: Mariana Grokes Marques. Brazilian Agricultural Research Corporation. Embrapa Swine and Poultry, BR-153, Km 110, Tamandua District, Postal Box: 321 CEP: 89715–899, Concórdia, SC. Brazil. Tel: +55 493441 0400. Fax: +55 493441 0497. E-mail: [email protected]
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Summary

The aim of this study was to optimize protocols for electroporation (EP) and polyfection (PLF) using polyethyleneimine (PEI) for pig sperm transfection and to determine which method was the most efficient. For EP standardization, different voltages, amounts and times of electric pulses were tested using propidium iodide (PI) as reporter. For PLF standardization, different concentrations of fluorescein isothiocyanate (FITC)-labelled PEI (PEI/FITC) were incubated with sperm for different periods of time. Flow cytometry was performed to evaluate the best protocol in terms of cell viability, including cytoplasmic membrane, acrosome, chromatin integrities and mitochondrial potential using the FITC probe, PI, acridine orange (AO) and JC1. Transfections with the plasmid pmhyGENIE-5 were carried out under optimum conditions for each procedure (EP: 500 volts, 500 μs and two pulses; PLF: PEI 0.5 mg/ml and incubation time 10 min). Transfection efficacy was assessed by fluorescence in situ hybridization (FISH). A lower transfection rate was observed for sperm in the control group (17.8%) compared with EP (36.7%), with PLF (76.8%) being the most efficient. These results suggest that the EP and PEI could be an efficient and low cost transfection method for swine sperm. Notably, treated cells showed higher plasmatic the membrane damage (PMD) and/or acrosome damage (AD) indexes, therefore the combination of this procedure with biotechniques that facilitate fecundation (i.e. in vitro fertilization or intracytoplasmic sperm injection) or even inclusion of antioxidant or anti-apoptotic drugs to improve spermatozoa viability would be important.

Keywords

ElectroporationFlow cytometryPigPolyethyleneimineSperm viability
Type
Research Article
Information
Zygote , Volume 26 , Issue 4 , August 2018 , pp. 286 - 293
Copyright
© Cambridge University Press 2018 

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